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`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 2 of 18 PageID #:
`9926
`AUGUST 1997
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`·t -LI N --I-CAL ·
`l~ -N -C-OLOGY
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`Univ. ·of ·Minn.
`Bio-Medical
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`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 3 of 18 PageID #:
`9927
`JOURNAL OF CLINICAL ONCOLOGY
`The Official Journal of the American Society of Clinical Oncology
`
`Journal of Clinical Oncology (ISSN 0732-J83X) is published monthly by the W.B. Saunders Company. Corporate
`and Editorial Office : The Curtis Center Independence Square West, Philadelphia, PA 19106-3399. Accounting and
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`Ameri an Society of Clinical Oncology-related questions should be addressed to ASCO, 225 Reinekers Lane,
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`Copyright © 1997 by American Society of Clinical Oncology. AJJ rights reserved. No part of this publication may
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`A Division of Harcourt Brace & Company
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`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 4 of 18 PageID #:
`9928
`
`Journal of Clinical Oncology
`
`The Official Journal of the American Society of Clinical Oncology
`
`Vol 15, No 8
`
`August 1997
`
`CONTENTS
`
`EDITORIAL: Chemotherapy Dose Escalation: Case Unproven . ... ... . .. Lillian L. Siu and Ian F. Tannock 2765
`
`Pediatric Oncology
`
`ORIGINAL REPORTS
`
`Randomized Study of Intensive MOPP-ABVD With or Without Low-Dose Total-Nodal Radiation Therapy
`in the Treatment of Stages IIB, IIIA2, IIIB, and IV Hodgkin's Disease in Pediatric Patients: A
`Pediatric Oncology Group Study ............ Michael A. Weiner, Brigid Leventhal, Martin L. Brecher,
`Robert B. Marcus, Alan Cantor, Peter W. Gieser, Jessie L. Temberg, Fred G. Behm, Moody D. Wharam, Jr,
`and Allen R. Chauvenet 2769
`
`Phase 1/11 Study of Idarubicin Given With Continuous Infusion Fludarabine Followed by Continuous
`Infusion Cytarabine in Children With Acute Leukemia: A Report From the Children's Cancer Group
`Patricia A. Dinndorf, Vassilios I. Avramis, Susan Wiersma, Mark D. Krailo, Wen Liu-Mares, Nita L. Seibel,
`Judith K. Sato, Revonda B. Mosher, John F. Kelleher, and Gregory H. Reaman 2780
`
`Role of Cranial Radiotherapy for Childhood T-Cell Acute Lymphoblastic Leukemia With High WBC Count
`and Good Response to Prednisone . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conter, M. Schrappe, M. Arico,
`A. Reiter, C. Rizzari, M. Dordelmann, M.G. Valsecchi, M. Zimmermann, W.-D. Ludwig, G. Basso, G. Masera,
`and H. Riehm for the Associazione ltaliana Ematologia Oncologia Pediatrica and the
`Berlin-Franlifurt-Munster Groups 2786
`
`Low-Grade Astrocytoma: A Decade of Experience at St. Jude Children's Research Hospital
`Amar Gajjar, Robert A. Sanford, Richard Heideman, Jesse J. Jenkins, Andrew Walter, Yulan Li,
`James W. Langston, Michael Muhlbauer, James M. Boyett, and Larry E. Kun
`
`Elevation of Homocysteine and Excitatory Amino Acid Neurotransmitters in the CSF of Children Who
`Receive Methotrexate for the Treatment of Cancer
`. . . . . . . . . . . . Charles T. Quinn, James C. Griener,
`Teodoro Bottiglieri, Keith Hyland, Arleen Farrow, and Barton A. Kamen
`
`2792
`
`2800
`
`Journal of Clinical Oncology (ISSN 0732-183X) is published monthly by W.B. Saunders Company. Corporate and Editorial Offices: The
`Curtis Center, Independence Square West, Philadelphia, PA 19106-3399. Accounting and Circulation Offices: W.B. Saunders Company,
`6277 Sea Harbor Dr, Orlando, FL 32887-4800. Periodicals postage paid at Orlando, FL 32862, and at additional mailing offices.
`Editorial correspondence should be addressed to George P. Canellas, MD, Journal of Clinical Oncology, 850 Boylston St, Suite 301A,
`Chestnut Hill, MA 02167. Telephone: (617) 739-8909. Fax (617) 739-8541. Email: whippend@jco.asco.org. Internet: http://
`www .jcojoumal.org/
`POSTMASTER: Send change of address to Journal of Clinical Oncology, clo W.B. Saunders Company, 6277 Sea Harbor Dr, Orlando,
`FL 32887-4800.
`Yearly subscription rates: United States and possessions: individual, $201.00; institution, $258.00; single issue, $26.00. All other countries:
`individual, $262.00; institution, $307 .00; single issue, $26.00. Student and resident: United States and possessions: $76.00, all other countries:
`$88.00. To receive student/resident rate, orders must be accompanied by name of affiliated institution, date of term, and the signature of
`program/residency coordinator on institution letterhead. Orders will be billed at individual rate until proof of status is received. Current
`prices are in effect for back volumes and back issues. Back issues sold in conjunction with a subscription are on a prorated basis. Subscriptions
`are accepted on a calendar year basis. Prices are subject to change without notice. Single issues, both current and back, exist in limited
`quantities and are offered for sale subject to availability.
`
`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 5 of 18 PageID #:
`9929
`
`Detection of Metastatic Neuroblastoma in Bone Marrow: When Is Routine Marrow Histology Insensitive?
`Nai-Kong V. Cheung, Glenn Heller, Brian H. Kushner, Chongyuan Liu, and Irene Y. Cheung
`
`2807
`
`Melanoma/ Sarcoma
`
`Detection of Circulating Melanoma Cells by Specific Amplification of Tyrosinase Complementary DNA Is
`Not a Reliable Tumor Marker in Melanoma Patients: A Clinical Two-Center Study
`Regine Glaser, Knuth Rass, Simone Seiter, Axel Hauschild, Enno Christophers, and Wolfgang Tilgen
`
`Evaluation of Tyrosinase mRNA as a Tumor Marker in the Blood of Melanoma Patients .............. .
`Fabio Alexandre Jung, Antonio Carlos Buzaid, Merrick Ira Ross, Kendra Victoria Woods, J. Jack Lee,
`Maher Albitar, and Elizabeth Ann Grimm
`
`Prognostic Factors Associated With Long-Term Surviva.l for Retroperitoneal Sarcoma: Implications for
`Management .... . . . ... ....... ... Martin J. Heslin, Jonathan J. Lewis, Evan Nadler, Efliot Newman,
`James M. Woodruff, Ephraim S. Casper, Denis Leung, and Murray F. Bren11a11
`
`2818
`
`2826
`
`2832
`
`Lung Cancer
`
`Randomized Trial of Alternating Versus Sequential Radiotherapy/Chemotherapy in Limited-Disease
`Patients With Small-Cell Lung Cancer: A European Organization for Research and Treatment of
`Cancer Lung Cancer Cooperative Group Study . . . . . . . . . . A1111a Gregor, Peter Drings, Jos Burglwuts,
`Piet E. Postmus, David Morgan, Tarek Sahmoud, Ann Kirkpatrick, Otilia Dalesio, and Giuseppe Giaccone
`
`2840
`
`Effect of Amifostine on Toxicities Associated With Sequential Chemotherapy and Radiation Therapy for
`Unresectable Non-Small-Cell Lung Cancer: Results of a Phase Il Trial
`. ... ... .. . Scott P. Ta1111ehill,
`Minesh P. Mehta. Marilyn Larson, Ban)• Srorer, John Pellet, Timothy J. Kinselfa, and Joan H. Schiffer 2850
`
`Immunocytochemical Markers in Stage I Lung Cancer: Relevance to Progno is . . . . . . . . . . Ugo Pastorino,
`Salvatore Andreola, Elda Tagliabue, Francesco Peu.clla, Matteo /11carbo11e, Gabriella Sozzi, Marc Buyse,
`Sylvie Menard, Marco Pierotti, and Franco Rilke
`
`2858
`
`Anti-Hu Antibodies in Patients With Small-Cell Lung Cancer: Association With Complete Response to
`Therapy and hnproved Survival ..... .. . ... . .... F. Graus, J. Dalmau, R. Rene, M. Tora, N. Malats,
`J.J. Verschuuren, F. Cardenal, N. Vifiolas, J. Garcia del Muro, C. Vadell, W.P. Mason, R. Rosell,
`J.B. Posner, and F.X. Real
`
`2866
`
`Breast Cancer
`
`Breast Conservation and Prolonged Chemotherapy for Locally Advanced Breast Cancer: The University of
`Michigan Experience . . . . . . . . . . . . Sofia D. Merajve,~ Barbara L. Weber, Roben Cody, Dao.wen Zhang,
`Myla Strawdennan, Kathleen A. Calzone, Virginia LeClaire, Albert Levin, Jennifer Irani, Mark Ha/vie,
`David August, Max Wicha Allen Lichter, and Lori J. Pierce
`
`2873
`
`High-Dose Chemotherapy and Stem-Cell Rescue in the Treatment of High-Risk Breast Cancer: Prognostic
`Indicators of Progression-Free and Overall Survival
`. . .......... George Somlo, James H. Doroshow,
`Stephen J. Fonnan, Tamara Odom-Maryon, Jemiifer lee, Warrell Chow, Victor Hamasaki, Lucille Leong,
`Robert Morgan, Jr, Kim Margolin, James Raschko, Stephen Shibata, Merry Tete!, Yun Yen, Jean Simpson,
`and Arturo Molina
`
`HER-2/neu Gene Amplification Characterized by Fluorescence In Situ Hybridization: Poor Prognosis in
`Node-Negative Breast Carcinomas ......... . . . Michael F. Press, Leslie Bernstein, Patricia A. Thomas,
`Lorraine F. Meisner, Jian-Yuan Zhou, Yanling Ma, Gene Hung, Robert A. Robinson, Charles Harris,
`Adel El-Naggar, Dennis J. Slamon Richard N. Phillips, Jeffrey S. Ross, Sandra R. Wolman,
`and Kerry J. Flom
`
`2882
`
`2894
`
`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 6 of 18 PageID #:
`9930
`
`Gastrointestinal Cancer
`
`Phase II Trial of 9-Aminocamptothecin Administered as a 72-Hour Continuous Infusion in Metastatic
`Colorectal Carcinoma
`. . . . . . Richard Pazdur, Enrique Diaz-Canton, W. Perry Ballard, James E. Bradof.
`Suzanne Graham, Susan G. Arbuck, James L. Abbruzzese, and Rodger Winn
`
`2905
`
`Phase II Trial of Irinotecan in Patients With Metastatic Colorectal Carcinoma
`. . . . . . . . . . Henry C. Pitot,
`Donald B. Wender, Michael J. O'Connell, Georgene Schroeder, Richard M. Goldberg, Joseph Rubin,
`James A. Mailliard, James A. Knost, Chirantan Ghosh, Ron J. Kirschling, Ralph Levitt,
`and Harold E. Windschitl
`
`2910
`
`Combined Modality Therapy for Stage II and Stage III Pancreatic Carcinoma . . . . . . . Arvind G. Kamthan,
`John C. Morris, Jack Dalton, John P. Mandeli, Margaret R. Chesser, Dvora Leben, Avram Cooperman,
`and Howard W. Bruckner 2920
`
`Genitourinary Cancer
`
`Bicalutamide for Advanced Prostate Cancer: The Natural Versus Treated History of Disease
`Howard I. Scher, Christine Liebertz, W. Kevin Kelly, Madhu Mazumdar, Chris Brett, Lawrence Schwartz,
`Geert Kolvenbag, Lisa Shapiro, and Morton Schwartz 2928
`
`Brain Tumors
`
`Effective Chemotherapy for Advanced CNS Embryonal Tumors in Adults . .. .. .. E. Galanis, J.C. Buckner,
`P.J. Schomberg, J.E. Hammack, C. Raffel, and B. W. Scheithauer 2939
`
`Hematologic Oncology
`
`Elderly Patients With Aggressive Non-Hodgkin's Lymphoma: Disease Presentation, Response to Treatment,
`and Survival-A Groupe d'Etude des Lymphomes de I' Adulte Study on 453 Patients Older Than 69
`Years ..... . . . . . .. Yves Bastion, Jean-Yves Blay, Marine Divine, Pauline Brice, Dominique Bordessoule,
`Catherine Sebban, Michel Blanc, Herve Tilly, Pierre Lederlin, Eric Deconinck, Bruno Salles,
`Charles Dumontet, Josette Briere, and Bertrand Coiffier 2945
`
`Human Urinary Macrophage Colony-Stimulating Factor Reduces the Incidence and Duration of Febrile
`Neutropenia and Shortens the Period Required to Finish Three Courses of Intensive Consolidation
`Therapy in Acute Myeloid Leukemia: A Double-Blind Controlled Study . ... ...... .... Ryuzo Ohno,
`Shuichi Miyawaki, Kiyohiko Hatake, Kazutaka Kuriyama, Kenji Saito, Akihisa Kanamaru, Tohru Kobayashi,
`Yoshihisa Kodera, Kiyoshi Nishikawa, Shin Matsuda, Osamu Yamada, Eijiro Omoto, Hideo Takeyama,
`Koji Tsukuda, Norio Asou, Mitsune Tanimoto, Hiroko Shiozaki, Masao Tomonaga, Tohru Masaoka,
`Yasusada Miura, Fumimaro Takaku, Yasuo Ohashi, and .Kazuo Motoyoshi 2954
`
`Cancer-Related Complications
`Phase m Double-Blind Comparison of Dolasetron Mesylate and Ondansetron and an Evaluation of the Additive
`Role of Dexamethasone in the Prevention of Acute and Delayed Nausea and Vomiting Due to Moderately
`Emetogenic Chemotherapy ...... ... ... Wycliffe S. Lofters, Joseph L. Pater, Benny Zee, Ellen Dempsey,
`David Walde, Jean-Pierre Moquin, Kenneth Wilson, Paul Hoskins, Raymond M. Guevin, Shailendra Verma,
`Rudolph Navari, James E. Krook, John Hainsworth, Michael Palmer, and Christine Chin 2966
`
`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 7 of 18 PageID #:
`9931
`
`Supportive Care
`
`Phase III Placebo-Controlled Trial of Capsaicin Cream in the Management of Surgical Neuropathic Pain in
`Cancer Patients .. .... ... Neil Ellison, Charles L. Loprinzi, John Kugler, Alan K. Hatfield, Angela Miser,
`Jeff A. Sloan, Donald B. Wender, Kendrith M. Rowland, Roy Molina, Terrence L. Cascino, Allen M. Vukov,
`Harbhajan S. Dhaliwal, and Chirantan Ghosh
`
`2974
`
`Clinical Impact of Chemotherapy Dose Escalation in Patients With Hematologic Malignancies and Solid
`Tumors . . . . . . . . . . . . . . . . . . . . . . . . . Diane M.F. Savarese, Chung-cheng Hsieh, and F. Marc Stewart 2981
`
`REVIEW ARTICLE
`
`Clinical Practice Guidelines for the Treatment of Unresectable Non-Small-Cell Lung Cancer
`the American Society of Clinical Oncology
`
`2996
`
`ASCO SPECIAL ARTICLE
`
`CNS Metastases: One's Too Many
`
`. .. . .... . .... .. ...... ... .... Andrew Lekos and Michael J. Glantz
`
`3019
`
`DIAGNOSIS IN ONCOLOGY
`
`Correspondence
`
`3021
`
`SPECIAL DEPARTMENTS
`
`Comparing an Apple to a Fruit Salad
`. . . . . . . . Nelson Spector, Wolmar Pulcher/, Marcia Nucci, and Angelo Maiolino
`• In Reply ............ •. . . . . Elihu Estey, Peter F. Thall, Sherry Pierce, Hagop Kantarjian, and Michael Keating
`
`Hodgkin's Disease Failures: Incorporating Radiotherapy and High-Dose Treatment . ... H. Miles Prince, Andrew Wirth,
`and Max Wolf
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valeria Bon/ante, Simonetta Viviani, and Gianni Bonadonna
`
`• In Reply
`
`Prospective Randomized Studies Are Needed to Define the Role of Axillary Lymph Node Dissection in Primary Breast
`Cancer .. . ............... . ..... S.D. Costa, G. von Minckwitz, J.F.H. Gauwerky, M. Kaufmann, and I.I. Diel
`• In Reply
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bruce G. Haffty
`
`Can Prophylactic CNS Radiotherapy Be Omitted in High-Risk Childhood Acute Lymphoblastic Leukemia?
`W. Balwierz, J. Armata, S. Skoczen, and W. Strojny
`. . . . . . . . . . . . . . . . . . . . . . . . Naomi Winick, Arleen Farrow, George R. Buchanan, Barton A. Kamen,
`Jonathan J. Shuster, W. Paul Bowman, and Michael Borowitz
`
`• In Reply
`
`Cutaneous Melanoma, Staging System
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Larry Nathanson
`• In Reply
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antonio C. Buzaid
`
`Announcements
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`cv
`
`Information for Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxvii
`
`Current Abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`xxix
`
`Books Received . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . lxxxvii
`
`Internet Access . . ... . ...... .. . ... ..... . . ... .... .. ... . . . , .. ... ............... .. ... .. .. .
`JCO Homepage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . www.jcojoumal.org
`ASCO OnLine
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . www.asco.org
`
`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 8 of 18 PageID #:
`9932
`
`HER-2/neu Gene Amplification Characterized by
`Fluorescence In Situ Hybridization: Poor Prognosis in
`Node-Negative Breast Carcinomas
`
`By Michael F. Press, Leslie Bernstein, Patricia A. Thomas, Lorraine F. Meisner, Jian-Yuan Zhou, Yanling Ma,
`Gene Hung, Robert A. Robinson, Charles Harris, Adel EI-Naggar, Dennis J. Slamon, Richard N. Phillips,
`Jeffrey S. Ross, Sandra R. Wolman , and Kerry J. Flom
`
`Purpose: The HER· 2/ neu gene codes for a membrane
`receptor protein that is homologous, but distinct from the
`epidermal growth factor receptor. This investigation was
`performed to validate fluorescence in situ hybridization
`(FISH) as a sensitive and specific method for assessing
`HER-2/neu gene amplification in archival tissue and to
`test whether this alteration is associated with poor prog·
`nosis.
`Materials and Methods: HER-2/ neu gene amplifica(cid:173)
`tion was determined by FISH in 140 archival breast can·
`cers, previously characterized for gene amplification by
`Southern hybridization or dot-blot hybridization, and for
`gene expression by Northern hybridization, Western im(cid:173)
`munoblot, or immunohistochemistry. A separate c.ohort
`of 324 node-negative breast cancers was assessed for
`amplification by FISH to determine the utility of HER-2/
`neu gene amplification.
`Results: Relative to solid-matrix blotting procedures,
`FISH analysis of HER-2/neu gene amplification showed
`a sensitivity of 98% and a specificity of 1 00% in 140
`
`breast cancers. Among patients treated by surgery only,
`the relative risks (relative hazard) of early recurrence
`(recurrent disease within 24 months of diagnosis), recur(cid:173)
`rent disease (at any time), and disease-related death
`were statistically significantly associated with amplifi(cid:173)
`cation. The prognostic information contributed by HER·
`2/ neu amplification was independent of the other mark·
`ers studied.
`Conclusion: FISH was an alternative technique for de(cid:173)
`termining gene amplification and had some distinct ad(cid:173)
`vantages over Southern hybridization. Our results dem·
`onstrate that HER-2/ neu gene amplification
`in the
`absence of adjuvant therapy is an independent predictor
`of poor clinical outcome and is a stronger discriminant
`than tumor size. Women with small tumors that hod gene
`amplification were at increased risk of recurrence and
`disease-related death.
`J Clin Oncol 15:2894-2904. © 1997 by American So(cid:173)
`ciety of Clinical Oncology.
`
`A MPLIFICATION AND OVEREXPRESSION of the
`
`HER-2/neu proto-oncogenc has been de cribed in
`approximately 25% of breast, ovarian, endometrial,
`gastric, and salivary gland carcinomas. 1
`5 These alter(cid:173)
`·
`ations have been associated with an increased risk of
`recurrent disease or shorter overall survival in patients
`with each of these cancers. 1
`6 HER-2/neu gene amplifi(cid:173)
`•
`cation is routinely analyzed by Southern or slot-blot
`hybridization. Although these are well-established and
`accepted methods, they require relatively large speci(cid:173)
`mens, are subject to dilutional artifacts from inclusion
`
`of normal cells, and, because they depend on nonde(cid:173)
`graded DNA, results are normally obtained from frozen
`Li sue. Based on the need to analyze small, paraffin(cid:173)
`embedded tissues rapidly on a cell-by-cell basis, we
`investigated the use of fluorescence in situ hybridiza(cid:173)
`tion (FISH) as a method for analysis of HER-2/neu
`gene amplification u ing a eries of archival tumor
`specimens previou ly characterized molecul arly for
`HER-2/neu gene amplification and expre
`ion. Final ly,
`324 axillary
`lymph node-negative breast cancers
`treated with surgical resection at three different institu-
`
`From the Norris Comprehensive Cancer Center and Departments
`of Pnrhology and Prevt!lllivl! Medicine, University of Southern Cali(cid:173)
`forn ia School of Medicine; Department of Hemat0/ogy-O11cology,
`University of Cnl,fom ia Los Angeles, School of Medicine, Los
`Angeles, CA: Dep11nment of Pathology, University of Iowa Hospitals
`and Clinics, Iowa City, IA; Cytogenetics Section, State Laboratory
`of Hygiene, University of Wisconsin, Madison, WI; Department of
`f(ll/,ology, M.D. Anderson Cancer Center, University of Texas,
`ltorlS/011. TX: Oncor, Inc, Gaithersburg, MD; and Depanmelll of
`Pathology, Albany Medical College, Albany, NY.
`Submitted August 13, 1996: accepted April 20, 1997.
`Supported in part by the National Institute of Child Health and
`Human Development (N0J-HD-3-3175); the National Cancer Insti(cid:173)
`tute (CA48780, CA58197 and CA50589), Bethesda, MD; the Univer-
`
`sity of outhem Califomill, Breasr Cancer Resew·clr Program. Los
`A118ele.1·, CA; the Revlo11/U11iversiry of C11llfornit1 Los Angeles Wom•
`en 's Cancer Research Progmm; the University of Califomia Los
`Angeles/U11iversi1y of Somhem Cal1fom ia Bnuw Tumor Bank
`(DAMD/7-94-1-4214); 1/r Molecular Core LLlboraU11)' Faci/iries of
`the General CU11ical RcseC1rch Center (National l11s1i1111es of Het1lt/1
`NCRR GCRC MOJ RR-43); and Oncor, Inc, Gaithersburg, MD.
`The Oncor HER-2/neu DNA Probe is for investigational use only.
`Address reprint requests
`to Michael F. Press, MD, PhD,
`NOR5412, Norris Com11reltensive Cancer Center, University of
`Southern California School of Medicine, 1441 Eastlake Ave, Los
`Angeles, CA 90033; email villalob@lrsc. usc. edu.
`© 1997 by American Society of Clinical Oncology.
`0732-183Xl97/l 508-0011$3.00/0
`
`2894
`
`Journol of Clinical Oncology, Vol 15, No 8 (August), 1997: pp 2894-2904
`
`

`

`Case 1:18-cv-01363-CFC Document 82-13 Filed 03/22/19 Page 9 of 18 PageID #:
`9933
`
`HER- 2/neu AMPLIFICATION BY FISH
`
`2895
`
`ti ons were analyzed to confi rm that HER-2/neu gene
`ampl ificati on by FISH was a marker of poor prog nosis.
`
`MATERIALS AND METHODS
`
`This study was performed in two phases. Phase I was performed
`to validate FISH as a method capable of assessing amplification
`in paraffin-embedded tissue. In phase II, parnffin-embedded ti$SUe
`section from cqucn tially diagnosed retrospectively identified inva(cid:173)
`sive node-negative breast cancer patients were used to determine
`whether gene amplification, assayed by FISH, was prognostically
`useful.
`
`HTB 132; American Type Culture Collection, Rockville, MD)"'
`(Zhou and Press, unpublished data, July 1993).
`If a total of I 00 nuclei could be identified and scored for both
`HER-2/11eu and chromosome 17 centromere, the sample was in(cid:173)
`cluded in the validation study. Based on tb e analysis of these results,
`scoring fewer nuclei was acceptable for the clinical outcome phase
`of the study (see Results). A mean HER-2/11eu gene copy per tumor(cid:173)
`cell nucleus and a mean chromosome 17 ce ntromere count per tu(cid:173)
`mor-cell nucleus were determined for each preparation. Using
`criteria established for Southern hybridi zation, 1
`11 HER-2/neu gene
`·'·
`amplification was defin ed as a HER-2/neu-to-chroinosome 17 ratio
`greater than 2.0.
`
`Phose /: Validation of FISH Using Molecularly
`Characterized Breast Cancers
`Breast cancer specimens with known HER-2/11eu gene copy and
`expression levels were selected for study as archival ti ssue speci(cid:173)
`mens. Amplification had been previously determined by Southern
`blot using DNA extracted from frozen tumor specimens and expres(cid:173)
`sion had been determined by Northern hybridization, Western immu(cid:173)
`noblotting, and immunohistochemistry using total RNA, total pro(cid:173)
`tein, and histologic stctions, respectively, from frozen tumor tissue.'
`Amplification had been previously analyzed by slot-blot analysis
`mid ex pression by immunohistochemistry alone in 19 cases. 6 To
`minimize inclusion of cases that were mi sclassified due to dilutional
`ai·ti/"acts associated with solid-matrix blotting methods, samples se(cid:173)
`lected as representative of gene amplification were required to show
`both amplification and overexpression, while cases selected as repre(cid:173)
`sentative of nonamplified samples were required to show no gene
`amplification or overexpression. One hundred forty cases were in(cid:173)
`cluded as validation samples for this phase of the study, with 50
`showing both amplification and overexpression and 90 showing no
`amplification or overexpression by solid-matrix blotting.
`Paraffin-embedded tissue sections. The breast cancer specimens
`used for the validation phase of the study were contained in two
`multitumor paraffin-embedded tissue blocks. 7 These blocks, pre(cid:173)
`pared according to the methods of Battifora and Mehta" from paraf(cid:173)
`fin-embedded tissue blocks obtained from institutional archives, con(cid:173)
`tained small strips of each breast cancer specimen. This methodology
`permits the use of small quantities of reagents and, more importantly,
`assures equal exposure of each breast cancer to all of the reagents
`in the assay system.
`FISH. HER-2/neu gene copy level was determined in paraffin(cid:173)
`embedded tissue sections. Since the HER-Zlneu gene is located on
`chromosome 17,9 an alpha satellite (pericentromeric) DNA probe
`(Oncor, Inc, Gaithersburg, MD) for chromosome 17 was selected as
`an internal control for chromosomal aneuploidy. By comparing the
`11111nber of copies of these two chromosomal markers, aneuploidy
`of chromosome 17 was excluded as a source of increased HER-2/
`11 e 11 gene copy number. The alpha-satellite DNA was also used as
`an internal control to correct for differences that might arise due to
`tissue sectioning artifact in paraffin-embedded sections. FISH was
`performed as previously described.'·' The nuclei were routinely
`counterstained with 4' -6' -diamidino-2' -phenylindole (OAP!). Some
`cases were also counterstained with acridine orange. Staining was
`visualized with a Zeiss fluorescence microscope. SK-BR-3 human
`breast cancer cells, known to have HER-2/neu gene amplification,
`and MDA-MB-468 human breast cancer cells, known to lack HER-
`2/neu gene amplification, were used as control cells (HTB30 and
`
`Phase II: Clinical Study of Axillary Lymph Node(cid:173)
`Negative Breast Cancers
`
`A total of 382 archival breast cancer speci mens were identified
`in the records of the M.D. Anderson Cancer Center ( 107 cases), the
`University of Wiscon si n (116 cases), and the Uni versity oflowa (159
`cases), and retrieved for analysis by FISH to assess the relationship
`between HER-2/neu gene amplification and three clinical outcomes.
`Inclusion in this study was based on a diagnosis of invasive breast
`cancer made before June I, 1990, lack of tumor in axillary lymph
`nodes, availability of tissue blocks, primary treatment by surgery
`alone without radiation therapy, adjuvant chemotherapy, or hormone
`therapy except after deve lopment of recurrent disease, and avai labil(cid:173)
`ity of at least 24 months of clinical follow-up information unless
`recurrent disease was identified. Cases were disqualified for lack of
`tumor or insufficient tissue in blocks (nine cases), failure to locate
`paraffini zed tissue blocks in the institutional archives (eight), a tech(cid:173)
`nically inadequate test result (two), ineligibl